Engineered hydrogel with aptamer structure confinement as well as preparation method and application of engineered hydrogel
By confined domain aptamers in the hydrogel network and controlling their anchor positioning and charge characteristics, an engineered hydrogel fluorescent probe with confined domains is constructed, which solves the identification of targets in complex environments by nucleic acid chains. The problem of performance interference achieves efficient identification and anti-interference capabilities.
Patent Information
- Application Number
- CN202411591514.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-02-18
AI Technical Summary
The adaptive folding behavior of the nucleic acid chain is susceptible to interference from various factors such as environmental pH, ionic strength, solvent polarity, and nuclease, which seriously weakens its performance in identifying binding targets in complex sample matrix.
A three-dimensional hydrogel network is used to limit the aptamers with the best target conformation, and the number of aptamers anchor anchors and relative positions, charge characteristics of polymerized monomers are engineered to construct an adaptation Engineered hydrogel fluorescent probes with domains of body structure.
The aptamer has achieved excellent affinity, recognition rate and excellent anti-interference ability in complex environments, significantly improving its performance in identifying heavy metal ions.
Smart Images
Figure CN119463019A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to an engineered hydrogel with a confined aptamer structure, a preparation method and application thereof, and belongs to the technical field of polymer intelligent materials. Background Art
[0002] Nucleic acid aptamers screened by in vitro exponential enrichment evolution technology have good chemical stability, high affinity, strong specificity, easy modification and large-scale preparation and other technical advantages. In recent years, they have been widely used in food safety, environmental monitoring, medical diagnosis and treatment and other fields. However, due to the inherent flexibility of oligonucleotide chains, the screening environment of aptamers is inconsistent with the application environment, resulting in the adaptive folding behavior of nucleic acid chains being easily interfered by environmental pH, ionic strength, solvent polarity, nucleases and other factors during the specific binding process with the target, which seriously weakens its ability to identify and bind targets in complex sample matrices.
[0003] Therefore, there is an urgent need in the art for an engineered hydrogel with a confined aptamer structure that can accurately identify a target, as well as a preparation method and application thereof. Summary of the invention
[0004] The purpose of the present invention is to solve the problem in the prior art that the adaptive folding behavior of nucleic acid chains is easily interfered by various factors such as environmental pH, ionic strength, solvent polarity, nucleases, etc., which seriously weakens its performance in identifying and binding targets in complex sample matrices.
[0005] In order to achieve the purpose of solving the above-mentioned problems, the technical solution adopted by the present invention is to provide an engineered hydrogel with a confined aptamer structure that can accurately identify the target, and a preparation method and application thereof.
[0006] In a first aspect of the present invention, a method for preparing an engineered hydrogel with a confined aptamer structure is provided, comprising the following steps:
[0007] Step 1, dissolving a heavy metal ion aptamer dry powder having a sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 8 in ultrapure water, annealing the powder in a metal bath, and then gradually cooling the powder to room temperature to obtain a heavy metal ion aptamer dry powder solution;
[0008] Step 2, mixing the heavy metal ion aptamer dry powder solution obtained in step 1 with the heavy metal ion aptamer solution in a buffer solution containing heavy metal ions, and incubating to obtain a heavy metal ion-aptamer complex solution;
[0009] Step 3, respectively adding NIPAA, MBAm and a positively charged monomer or a negatively charged monomer to the heavy metal ion-aptamer complex solution obtained in step 2; introducing nitrogen gas to completely remove oxygen, and incubating at 4°C to fully form a prepolymer mixture; adding SDS, APS and TEMED to the above mixture to initiate polymerization, adding 10 mM HCl after the polymerization to elute the heavy metal ions, and eluting and removing HCl with deionized water to obtain an engineered hydrogel with a confined aptamer structure, and storing at 25°C for future use.
[0010] Wherein, the sequence of the heavy metal ion aptamer dry powder is shown in the following table:
[0011]
[0012] Preferably, in step 1, the metal bath temperature is 95° C. and the annealing time is 5 min.
[0013] Preferably, in step 2, the amount of heavy metal ion solution added is 25-60 μL, 10 mM; the amount of heavy metal ion aptamer is 10-40 μL, 100 μM; the amount of buffer containing heavy metal ions is 20 mM, pH 5.5, and the total volume is 588 μL; the incubation temperature is 37° C., and the time is 30 min.
[0014] Preferably, in the step 3, the amount of NIPAA added is 20-42 μL, 1 M; the amount of MBAm added is 160-240 μL, 0.1 M; the amount of the positively charged monomer or the negatively charged monomer added is 5-15% of the monomer ratio of the gel; the positively charged monomer is selected from N-[3-(dimethylamino)propyl]methacrylamide (DMPA), acrylamide (AAm) and 3-(acrylamidopropyl)trimethylammonium chloride (AAPTAC); the negatively charged monomer is selected from 2-acrylamide-2-methylpropanesulfonic acid (APMS), acrylic acid (AAc), sodium p-vinylbenzenesulfonate (SS) and sodium propylenesulfonate (AS).
[0015] Preferably, in step 3, the amount of the positively charged monomer DMAP is 10, 8, or 6 μmol, and the amount of the negatively charged monomer AMPS is 10 μmol.
[0016] The second aspect of the present invention provides an engineered hydrogel with confined aptamer structure prepared by the above method.
[0017] The third aspect of the present invention provides an application of an engineered hydrogel with a confined aptamer structure prepared by the above method in identifying heavy metal ions, comprising the following steps:
[0018] Step 1: Incubate the engineered hydrogel with the aptamer structure confined with lanthanide aqueous solution and buffer (20mM MES pH 5.5) with shaking to construct the heavy metal ion aptamer optimal conformation confined hydrogel fluorescent probe (Acrydite-TH@Ln 3+ );
[0019] Step 2: Mix the fluorescent probe obtained in step 1 with a buffer solution containing heavy metal ions, incubate, monitor the fluorescence intensity at 548 nm, and establish a linear relationship between different heavy metal ion concentrations and fluorescence intensity.
[0020] Preferably, in step 1, the amount of the engineered hydrogel with confined aptamer structure is 1 mL 2-10 mg / L; the amount of the lanthanide aqueous solution is 0.02 mL 5-40 mM; the incubation conditions are: 25°C, 1000 rpm, incubation time 15-60 min, and the amount of buffer is 0.2-0.5 mL.
[0021] Preferably, in step 2, the heavy metal ion is Pb 2+ or Cd 2+ The amount of buffer containing heavy metal ions is 0.2 to 0.5 mL.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] 1. The aptamer with the most suitable conformation for target recognition was confined by a three-dimensional hydrogel network, and the number and relative position of the aptamer anchoring sites, the charge characteristics of the polymerized monomers and other parameters were engineered and regulated. The three-site co-anchored optimal conformation confined positively charged hydrogel type III fluorescent probe (5'-T8-3'TH(III)@Tb 3+ ) showed excellent affinity, recognition rate and outstanding anti-interference ability.
[0024] 2. A label-free fluorescent probe was constructed by utilizing the fluorescence sensitization of oligonucleotide molecules to lanthanide metals and the low optical background characteristics of hydrogels.
[0025] 3. The engineered hydrogel fluorescent probe with confined aptamer structure can be expanded to detect other heavy metal ions. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 The optimal conformational domain for the three-site co-anchoring of positively charged hydrogel type III fluorescent probe (5'-T8-3'TH(III)@Tb 3+ ) Construction and Pb 2+ Detection feasibility, A: 5'-T8-3'TH(III)@Tb 3+ Feasibility diagram of fluorescent probe construction, B: 5'-T8-3'TH(III)@Tb3+ Feasibility diagram of fluorescent probe sensing.
[0027] Figure 2 The influence of the number and relative position of the anchoring sites of the aptamer in the hydrogel on the recognition affinity is studied for the optimal recognition conformation.
[0028] Figure 3 The effect of the charge characteristics of hydrogel polymer monomers on the recognition rate. A: The positive and negative charge aptamer confined hydrogel on Pb 2+ Adsorption kinetics study, B: Adsorption of Pb by aptamer-confined hydrogels with different positive charge contents 2+ Adsorption kinetics studies.
[0029] Figure 4 5'-T8-3'TH(III)@Tb 3+ Fluorescent probe detection of Pb 2+ Standard curve and selectivity, A:Pb 2+ Detection spectrum, B: Pb 2+ Detection linear fitting results, Figure C, selectivity.
[0030] Figure 5 5'-T8-3'TH(III)@Tb 3+ Study on the anti-interference ability of fluorescent probes, A: ionic strength (Na + ), B: pH, C: nucleic acid denaturant (MeOH), D: nuclease (exonuclease I).
[0031] Figure 6 For general use - Cd 2+ Establishment and selectivity of detection method, A: Cd 2+ Detection spectrum, B: Cd 2+ Detection linear equation fitting results, C: selectivity. DETAILED DESCRIPTION
[0032] In order to make the present invention more clearly understood, a preferred embodiment is described in detail with reference to the accompanying drawings as follows:
[0033] like Figure 1-6 As shown, the present invention provides an engineered hydrogel with confined aptamer structure, a preparation method and application thereof.
[0034] Example 1
[0035] 1) Lead ions induce nucleic acids to form the best recognition conformation
[0036] The heavy metal ion aptamer dry powder shown in the sequence of SEQ ID No. 1 in the table was dissolved in ultrapure water, annealed at 95°C for 5 minutes, and cooled to room temperature. 40 μL of 100 μM lead ion aptamer solution was added to MES buffer (20 mM, pH 5.5) containing 250 nmol lead ions, and incubated at 37°C for 30 minutes to prepare the lead ion-aptamer complex.
[0037] 2) Preparation of engineered hydrogels with confined lead ion aptamers
[0038] 1.5-3 mg AAm, 2-5 mg NIPAA, DMPA (0.3-0.9 μL) and 1.5-3.5 mg MBAAm were added to the above solution respectively, and the final volume was 1 mL. Nitrogen was introduced for 0.5 h to remove oxygen from the solution, and the solution was incubated at 4 ° C for 8 h to form a prepolymer. Subsequently, 20-40 μL 60 mg / mL SDS, 20-60 μL 100 mg / mL APS and 0.3-0.9 μL TEMED were added to initiate polymerization, and the polymerization was carried out at room temperature for 12 h. The product was obtained by centrifugation; 10 mM HCl was added to elute Pb 2+ , and then eluted with deionized water to remove HCl and stored at 25°C for later use to obtain an engineered hydrogel with a confined lead ion aptamer structure.
[0039] Application Example 1: Establishment of Pb based on fluorescent probe 2+ Detection Methods
[0040] 200 μL of 10-60 mM lanthanide (Ln 3+ ) aqueous solution and 1 mL of MES buffer (20 mM, pH 5.5), incubate at 25°C, 1000 rpm, shake for 15 to 60 min. Take 50 to 100 μL of the above fluorescent probe solution and add 200 to 400 μL of different concentrations of Pb 2+ MES buffer solution, 25°C, 1000 rpm, incubate for 15 min with shaking, and record the fluorescence intensity at 548 nm.
[0041] Application Example 2: Verification of Universality
[0042] The heavy metal ion aptamer dry powder shown in SEQ ID No. 8 in the table was dissolved in ultrapure water, annealed at 95°C for 5 min, and cooled to room temperature. 40 μL of 100 μM cadmium ion aptamer solution was added to a 250 nmol Cd 2+The cadmium ion-aptamer complex was prepared by incubating at 37°C for 30 min in HEPES buffer (10 mM, pH 7.0). 3 mg AAm, 5 mg NIPAA, DMPA (0.6 μL) and 22.5 mg MBAAm were added to the above solution, respectively, and the final volume was 1 mL. Nitrogen was introduced for 0.5 h to remove oxygen from the solution, and the solution was incubated at 4°C for 8 h to form a prepolymer. Subsequently, 20 μL 60 mg / mL SDS, 20 μL 100 mg / mL APS and 0.3 μL TEMED were added to initiate polymerization, and polymerization was carried out at room temperature for 12 h. The product was obtained by centrifugation and labeled as 5'-21Cd 2+ Apt-Hydrogel@Tb 3+ . Add 10 mM HCl to elute Cd 2+ , and then eluted with deionized water to remove HCl and stored at 25°C for future use. The probe construction and detection steps are the same as those described above for Pb 2+ Consistent.
[0043] Figure 1 5'-T8-3'TH(III)@Tb 3+ Fluorescence sensor construction and Pb 2+ Feasibility of detection. Figure 1 As shown in .A, with Tb 3+ Add to 5'-T8-3'TH(III), 5'-T8-3'TH(III)@Tb 3+ It showed excellent fluorescence signal at 548 nm, while 5'-T8-3'TH(III) and Tb 3+ There is no obvious fluorescence signal, which indicates that 5'-T8-3'TH(III)@Tb 3+ The fluorescent probe was successfully constructed; with different concentrations of Pb 2+ , 5'-TH@Tb 3 The fluorescence intensity showed a gradient decrease, which indicated that 5'-T8-3'TH(III)@Tb 3+ Detection of Pb 2+ feasibility( Figure 1 .B).
[0044] Figure 2 The results show that the optimal conformation of the aptamer has an influence on the number and relative position of the hydrogel anchoring sites on Pb 2+ The influence of affinity. Figure 2 As shown in the figure, under the single-site fixation method, 5', 3', and T8 showed better Pb 2+ Better affinity. In the multi-site fixation mode, the three sites co-anchor the optimal conformational domain positively charged hydrogel type III fluorescent probe (5'-T8-3'-TH(III)@Tb3+ ), and obtained the 2+ The best affinity, Kd, was 0.97 μM, corresponding to the aptamer hydrogel fluorescent probe with the same sequence and non-G4 conformation co-fixed at three sites (Control W / O 5'-T8-3'-TH@Tb 3+ , W / O is the control and does not form G4 conformation, and the sequence is completely consistent with SEQ ID NO.7. ) The affinity Kd is 3.2M, which is nearly 12.8 times higher than that of the single-site fixation method.
[0045] Figure 3 It is shown that the charge characteristics of hydrogel polymer monomers have an influence on the Pb 2+ The effect of binding rate. Figure 3 As shown in A, the three sites co-anchored the hydrogel fluorescent probe with different charge characteristics in the optimal conformational quadrant (positive charge: 5'-T8-3'TH(I)@Tb 3+ and negative charge: 5'-T8-3'TH(II)@Tb 3+ ) on Pb adsorption kinetics experiments showed that aptamer-functionalized hydrogels with different charge characteristics had a strong adsorption capacity for Pb 2+ The adsorption behavior is more consistent with pseudo-second-order kinetics (pseudo-second-order kinetics R 2 Both are greater than pseudo first-order R 2 ), which indicates that chemical adsorption is dominant in the adsorption process. 3+ 5'-T8-3'TH(I)@Tb 3+ It also shows better binding rate and binding effect. To this end, this patent explores the effect of different charge densities on Pb 2+ The effect of adsorption rate ( Figure 3 .B), DMPA was used as a positively charged monomer, and aptamer functionalized hydrogels with different positive charge densities were prepared by adjusting its addition amount. When the DMPA content was too high, the gel layer was too thick, resulting in 5'-T8-3'TH(II)@Tb 3+ The adsorption rate decreased. When the DMPA content was too low, the optimal conformation of the aptamer could not be completely fixed, which led to poor binding effect. When the DMPA content was 8 μmol, 5'-T8-3'TH(III)@Tb 3+ Shows the best combination effect.
[0046] Figure 3 The experimental data are shown in Table 1:
[0047] Table 1 Fitting parameters of adsorption kinetics of aptamer confined hydrogels with different charge characteristics
[0048]
[0049]
[0050] a:F 0 / F-1 ,exp is the experimental value of F 0 / F-1.
[0051] b:F 0 / F-1 ,cal is the calculated value of F 0 / F-1.
[0052] In Table 1, 5'-T8-3'-TH(I)@Tb 3+ Representative of the three-site co-anchored optimal conformational domain negatively charged hydrogel (I) type fluorescent probe, 5'-T8-3'-TH(II)@Tb formed by adding 10 μmol APMS 3+ 、5'-T8-3'-TH(III)@Tb 3+ and 5'-T8-3'-TH(IV)@Tb 3+ Represents the formation of three-site co-anchored optimal conformational domain positively charged hydrogel (II), (III) and (IV) type fluorescent probes by adding 10 μmol, 8 μmol and 6 μmol DMPA in sequence.
[0053] Figure 4 It is shown that 5'-T8-3'TH(III)@Tb 3+ Detection of Pb 2+ Standard curve and selectivity. Figure 4 As shown in A and B, in the range of 0.005-80 μM, 5'-T8-3'TH(III)@Tb 3+ Fluorescence intensity and Pb 2+ The concentration showed a good linear relationship, and the detection limit LOD was 1.52nM. Figure 4 .C indicates 5'-T8-3'TH(III)@Tb 3+ Pb 2+ Has good selectivity.
[0054] Figure 5 The effect of the present invention on the anti-interference ability of aptamers was studied. Figure 5 AD, respectively explored the ionic strength (Na + ), pH, nucleic acid denaturant (methanol), exonuclease I and other interfering environments have an impact on the 5'-T8-3'TH(III)@Tb 3+ Detection of Pb 2+ The effects of free lead ion aptamer (T30596) and lanthanide elements (Tb 3+) constructed fluorescent probe (T30695@Tb 3+ ) In comparison, the strategy of anchoring the aptamer's optimal recognition conformation in the hydrogel significantly improves the aptamer's anti-interference ability.
[0055] Figure 6 Display, Pb 2+ The aptamer sequence was replaced with Cd 2+ After the aptamer, Figure 6 A and B, 5'-21Cd 2+ Apt-Hydrogel@Tb 3+ Cd 2+ It shows good detection effect, such as Figure 6 C, 5'-21Cd Apt-Hydrogel@Tb 3+ Demonstrates excellent selectivity.
[0056] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and equivalent changes made by using the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. A method for preparing an engineered hydrogel with a confined aptamer structure, characterized in that: The following steps are involved: Step 1, dissolving a heavy metal ion aptamer dry powder having a sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 8 in ultrapure water, annealing the powder in a metal bath, and then gradually cooling the powder to room temperature to obtain a heavy metal ion aptamer dry powder solution; Step 2, mixing the heavy metal ion aptamer dry powder solution obtained in step 1 with the heavy metal ion aptamer solution in a buffer solution containing heavy metal ions, and incubating to obtain a heavy metal ion-aptamer complex solution; Step 3, respectively adding NIPAA, MBAm and a positively charged monomer or a negatively charged monomer to the heavy metal ion-aptamer complex solution obtained in step 2; introducing nitrogen gas to completely remove oxygen, and incubating at 4°C to fully form a prepolymer mixture; adding SDS, APS and TEMED to the above mixture to initiate polymerization, adding 10 mM HCl to elute the heavy metal ions after the polymerization, and eluting and removing HCl with deionized water to obtain an engineered hydrogel with a confined aptamer structure, and storing it at 25°C for future use.
2. The method for preparing the engineered hydrogel with confined aptamer structure according to claim 1, characterized in that: In the step 1, the metal bath temperature is 95° C. and the annealing time is 5 min.
3. The method for preparing the engineered hydrogel with confined aptamer structure according to claim 1, characterized in that: In the step 2, the amount of heavy metal ion solution added is 25-60 μL, 10 mM; the amount of heavy metal ion aptamer is 10-40 μL, 100 μM; the amount of buffer solution containing heavy metal ions is 20 mM, pH 5.5, and the total volume is 588 μL; the incubation temperature is 37° C., and the time is 30 min.
4. The method for preparing the engineered hydrogel with confined aptamer structure according to claim 1, characterized in that: In the step 3, the amount of NIPAA added is 20 (42 μL, 1 M; the amount of MBAAm added is 160 (240 μL, 0.1 M; the amount of positively charged monomer or negatively charged monomer added is 5-15% monomer ratio of the gel; The monomer with positive charge is selected from N-[3-(dimethylamino)propyl]methacrylamide, acrylamide and 3-(acrylamidopropyl)trimethylammonium chloride; the monomer with negative charge is selected from 2-acrylamide-2-methylpropanesulfonic acid, acrylic acid, sodium p-vinylbenzenesulfonate and sodium propylenesulfonate.
5. The method for preparing the engineered hydrogel with confined aptamer structure according to claim 1, characterized in that: In the step 3, the amount of the positively charged monomer DMAP is 10, 8, and 6 μmol, and the amount of the negatively charged monomer AMPS is 10 μmol.
6. An engineered hydrogel with confined aptamer structure prepared by any one of the methods of claims 1 to 4.
7. Use of the aptamer structure-confined engineered hydrogel as claimed in claim 6 in identifying heavy metal ions, characterized in that: The following steps are involved: Step 1: Incubate the engineered hydrogel with restricted aptamer structure with lanthanide aqueous solution and buffer solution under shaking to construct a hydrogel fluorescent probe with the optimal conformation of heavy metal ion aptamer restricted domain; Step 2: Mix the fluorescent probe obtained in step 1 with a buffer solution containing heavy metal ions, incubate, monitor the fluorescence intensity at 548 nm, and establish a linear relationship between different heavy metal ion concentrations and fluorescence intensity.
8. Use of the engineered hydrogel with restricted aptamer structure as claimed in claim 7 in identifying heavy metal ions, characterized in that: In the step 1, the amount of the engineered hydrogel with confined aptamer structure is 1 mL 2-10 mg / L; the amount of the lanthanide aqueous solution is 0.02 mL 5-40 mM; the incubation conditions are: 25° C., 1000 rpm, incubation time 15-60 min, and the amount of buffer is 0.2-0.5 mL.
9. Use of the engineered hydrogel with restricted aptamer structure as claimed in claim 7 in identifying heavy metal ions, characterized in that: In the step 2, the heavy metal ion is Pb 2+ or Cd 2+ The amount of buffer containing heavy metal ions is 0.2 to 0.5 mL.